Most human hemoglobins are composed of two pairs of unlike subunits which confer the molecule a unique property, i.e., conformational change during reaction (oxygenation). Although a great deal is known about structure-function relationship of hemoglobin, the mode of enzymatic reduction of methemoglobin is the least studied. The first part of this project deals with purification of NADH-methemoglobin reductase by affinity or iron exchange chromatography and the study of methemoglobin reduction and its dependence on conformational change. The second part deals with the effect of Cl minus and CO2 on the oxidation of hemoglobin subunits and their effect on the conformation of these subunits. Studies conducted during the last year consist of purification of methemoglobin reductase by ion-exchange chromatography up to about 4000-fold and the reduction of methemoglobin by this enzyme in the presence and absence of inosine hexaphosphate (IHP). It was shown that methemoglobin reduction with a rapid and a slow phase with 4-6 fold difference in their rate. Reoxidation of partially reduced samples by copper (Cu II) showed that the rapidly reacting component consisted of beta chains. When a completely different technique, i.e., reduction and chain separation by polyacrylamide gel electrophoresis was used, similar results were obtained. IHP in low concentration accelerates the reduction rates for both subunits but does not alter the preferential reduction of beta chains. It is concluded that as alpha chains autoxidize with a rate of 8-10 times faster than the beta chains (previously reported), there is at all times a larger pool of oxidized alpha chains. In order to offset that, the preferential reduction of beta subunits seems necessary.